On the road to CLL

PAOLO GHIA

Strategic Research Program on CLL - Lab of B Cell Neoplasia

Division of Experimental Oncology

Università Vita-Salute San Raffaele - Milano

IRCCS Istituto Scientifico San Raffaele - Milano

Low-count

MBL

CLL

GC

HSC CD5+ B cell

Primary event Genetic

alteration

Chronic

microenvironmental

(Antigenic?)

stimulation

Antigen

T

Th

APCs

Antigen-

primed

DC

Genetic

alteration

High-count

MBL

T cell-independent

A long path to CLL

Modified from Bertilaccio et al., Leukemia 2013

Genetic

alteration

Richter’s

Syndrome

Barrio. et al, EHA 2016 poster #194; Hernandez-Sanchez et al, EHA 2016, oral presentation #115

• Characteristic phenotype: CD5+, CD23+, CD20dim, sIgMdim

CD

5

CD

19

Kappa CD20 CD79b CD5

Kappa CD20 CD79b

CD

5

CD5

CD

19

Chronic Lymphocytic Leukemia (CLL)

0

1

2

3

4

5

6

7

8

9

10

<40 40-49 50-59 60-69 70-79 ≥80

Perc

en

tag

e (

%)

Years

Male Female

5.2% >18yy

Dagklis et al, Blood 2009

6.7% >40yy

CLL-like MBL is common among the elderly

Scarfò et al, Sem Canc Biol 2010

In individuals >40 years of age

The more you search, the more you find...

Nieto et al, Blood 2009

500.000 events

12% overall

5.000.000 events

Scarfò et al, Sem Canc Biol 2010

5.2% overall

Are we all bound to have it?

0

1

2

3

0 – 0.5 0.5 – 5.0 >5.0

Pre

va

len

ce

(%

)

B-Lymphocyte count (109/L)

High-count MBL CLL

Are all MBL equal?

Rawstron et al, NEJM 2009

Biological features of high-count MBL

0

1

2

3

0 – 1.0 0.5 – 5.0 >5.0

Pre

va

len

ce

(%

)

B-Lymphocyte count (109/L)

(General population)

Low-count MBL

CLL

1.1% per year CLL

(Clinical)

High-count MBL

Are all MBL equal?

Fazi, Scarfò et al, Blood 2011

MBL cells tend to persist with time

A

B

Fazi, Scarfò et al, Blood 2011

Median follow-up time= 34 months

MBL cells tend to remain stable with time

Low-count MBL

Normal B-cells

P=0.430

100

80

60

40

20

0 6 1 2 3 4 0 5

Perc

enta

ge a

live

Years since screening

CLL-like MBL: 5/48 oncology, 3/5 MBL Controls: 0/88 oncology referrals

CLL-like B-cells typically <0.01 x 109/L

A. Rawstron unpublished 2015

Del13q14.3

Trisomy 12

Fazi, Scarfò et al, Blood 2011

7/16 cases

43.8%

Del17p13

1/16 cases

6.3%

2/16 cases

12.5%

ID SEX AGE

Follow-up

(months)

Clone

(cells per μl)

FISH IGHV gene

IGHV

homology

1st visit

Follow-

up

VB0314 F 78 47 5,79 4,82 Del13q14.3*, Trisomy 12 IGHV4-59/-61 94,4

VB0588 F 65 39 96,73 319,10 Del13q14.3 IGHV4-59 93,5

VB1002 F 63 35 1,67 1,60 Del17p IGHV4-59/-61 97,0

VB1134 M 58 34 2,36 0,46 Del13q34 n.d. n.d.

VB1202 F 61 34 4,72 18,56 Del13q14.3 n.d. n.d.

VB1320 F 61 24 0,41 3,82 Del13q14.3-q34, Del17p n.d. n.d.

VB1377 M 68 24 3,78 2,42 Del13q34 n.d. n.d.

VB1470 F 73 23 0,94 4,02 Del13q14.3* n.d. n.d.

Low-count MBL carry identical genetic lesions

TP53 disruption is associated with poor prognosis

Wt: wildtype; OS: overall survival

Nonsense Missense Frameshift

5’ 3’

1

DNA BINDING

EX4 EX9

393 TP53

TP53 M

17p-

TP53 M /17p-

Wt

Aberration Incidence (%)1

Median OS (months)1

17p del 7 32

11q del 18 79

+12 16 114

Normal 18 111

13q del 55 133

13q deletion as sole abnormality

17p deletion

Normal Trisomy 12q 11q deletion

Months

% S

urv

ivin

g

0 12 24 36 48 60 72 84 98 108 132 156 180

100

80

40

20

0

60

Wt (n=277; median not reached)

TP53 M (n=14; 30.2 median months) 17p- (n=16; median 19.2 months)

Time (months)

Fra

ctio

n A

live

0 OS2

0 12 24 36 48 60 72 84 96 108

1.0

0.4

0.1 0.2 0.3

0.5 0.6

0.9 0.8 0.7

del13q14

del17p13 +12

del11q22-q23

OS1

1. Döhner H, et al. N Engl J Med 2000;343:1910–6; 2. Zenz T, et al. J Clin Oncol 2010;28:4473–9.

TP53 mutation and/or del(17p) predict non-response to first-line chemoimmunotherapy

O

S p

rop

ort

ion

0

0.4

0.6

0.8

1.0

0.2

0 96 90 84 78 72 66 60 54 48 42 36 30 18 12 6 24

Months

del(17p) del(11q) Trisomy 12 del(13q) Normal

FC FCR

O

S p

rop

ort

ion

0

0.4

0.6

0.8

1.0

0.2

0 96 90 84 78 72 66 60 54 48 42 36 30 18 12 6 24

Months

FC and TP53 WT FC and TP53 mut FCR and TP53 WT FCR and TP53 mut

FC, fludarabine, cyclophosphamide; FCR, FC, fludarabine, cyclophosphamide, rituximab; OS, overall survival Stilgenbauer S, et al. Blood 2014;123:3247–54

16

Fazi, Scarfò et al, Blood 2011

Del17p13 2/16 cases

12.5%

Patients 17p- may have stable disease

Best et al, Leukemia 2009

TP53 disruption is associated with poor prognosis

Aberration Incidence (%)1

Median OS (months)1

17p del 7 32

11q del 18 79

+12 16 114

Normal 18 111

13q del 55 133

13q deletion as sole abnormality

17p deletion

Normal Trisomy 12q 11q deletion

Months

% S

urv

ivin

g

0 12 24 36 48 60 72 84 98 108 132 156 180

100

80

40

20

0

60 del13q14

del17p13 +12

del11q22-q23

OS1

Dohner et al. NEJM 2000 Calin et al. PNAS 2002

Döhner, New Engl J Med 2000

Klein et al, Cancer cell, 2010

Del 13q in CLL and MBL

Cimmino, et al. (2005) PNAS

BCL2 is a target of miR-15 and miR-16

actin

BCL2

actin

Po

sit

ive c

on

tro

l

To

- - + 72h

Mcl1

Scielzo et al, Leukemia 2011, Fonte et al, Clin Can Res, 2013 Hanada M et al, Blood 1993, Kitada S et al, Blood 1998

BCL2 is overexpressed in CLL

+ 24h

Po

sit

ive c

on

tro

l

To

- - + 72h

+ 24h

actin

BCL2

CD40L

CD40L

Bcl-2

Burger, Ghia et al, Blood, 2009

Microenvironmental stimuli in CLL

Ballesteros et al, EHA 2016, poster #1037

Damle et al, 1999 Time from diagnosis

Hamblin et al, 1999

IG genes can be mutated in CLL

Herishanu Y et al. Blood 2011

BCR is activated in the LN microenvironment

BCR signalling in CLL is heterogeneous

Bad prognosis

Survival

Proliferation

Good prognosis

Zupo et al, 1996; Chen et al, 2002; Lanham et al, 2003

BCR reactivity in CLL: self and foreign antigens

Dühren-von Minden et al, Nature 2012 Hervé et al, JCI 2005, Lanemo Myhrinder et al, Blood 2008, Hoogeboom et al, JEM 2013,

Kostareli et al, Leukemia 2011; Hwang et al, PlosOne 2014; ten Hacken et al, EHA 2016, poster #195

• 30.4% of all CLL cases (2308/7596)

Agathangelidis et al, 2012 Blood

H

L

H

L HCDR3

FR2 FR3 FR1

CDR1 CDR2

IGHJ IGHV

Stereotyped receptors in CLL

Gounari et al, EHA 2016, oral presentation #116

Subset #4

Subset #2

Stereotyped subsets have a distinct clinical course

Baliakas et al. Lancet Haematology 2014 Baliakas et al. EHA 2016, poster #598

Baliakas et al. Lancet Haematology 2014

Poor clinical course is independent of cytogenetics

Subsets

Cu

mu

lati

ve

eve

nts

Strefford et al, Leukemia, 2013; Malcikova et al, BJH 2014

Subset 1 Subset 2 Subset 8

Rossi et al, Blood, 2013

Gene mutations and IG stereotypy

Genetic abnormalities

MBL overt CLL Refractory CLL/

Richter Syndrome Normal B cell

Low-count

MBL

CLL

GC

HSC CD5+ B cell

Primary event Genetic

alteration

Chronic

microenvironmental

(Antigenic?)

stimulation

Antigen

T

Th

APCs

Antigen-

primed

DC

Genetic

alteration

High-count

MBL

T cell-independent

A long path to CLL

Modified from Bertilaccio et al., Leukemia 2013

Genetic

alteration

Richter’s

Syndrome

Barrio. et al, EHA 2016 poster #194; Hernandez-Sanchez et al, EHA 2016, oral presentation #115

IRCCS Istituto Scientifico San Raffaele

Laboratory of B Cell Neoplasia

Lydia Scarfò, Alessandra Rovida, Engin Bojnik,

Costantinos Xhantopoulos, Silvia Heltai,

Pamela Ranghetti, Silvia Bonfiglio

Strategic Research Program on CLL

Lydia Scarfò, Piera Angelillo, Maria Colia, Antonella

Capasso, Virginia Sgarlato, Stefania Cresta, Eloise Scarano

Division of Experimental Oncology

CERTH, Thessaloniki

Anna Vardi, Stavroula Ntoufa,

Aliki Xochelli, Anastasia

Hadzidimitrious, Andreas

Agathangelidis, Maria Gounari,

Kostas Stamatopoulos

Karolinska Institut, Stockholm

Lesley Ann Sutton, Panayotis

Baliakas, Viktor Ljungstrom,

Richard Rosenquist